The Study On Repair Of Adult Rat Spinal Cord Injury By Recombinant TORC1 Lentivirus Vector

Partâ… : Construction and Identification of RecombinantLentivirus Carrying SD Rat TORC1 GeneObjective To construct a recombinant lentivirus which carried SD rat TORC1 (transducerof regulated CREB activity 1) gene and examine its ability to express the TORC1 gene invitro. Methods The coding sequence of SD rat TORC1 gene was amplified using PCR andcloned into pGC-FU vector. 293T cells were transfected using Lipofectamine 2000 andpackaged for the recombinant lentivirus particles. When the cloned sequence was identifiedto be right, the recombinant lentivirus particles were amplified in a large quantity. The titerof virus was determined by real-time PCR and the level of TORC1 expression wasexamined by Western blot. Results The recombinant lentivirus carrying TORC1 wasconstructed successfully and could express TORC1 at a high level in 293T in vitro.Conclusion The recombinant lentivirus could express TORC1 gene at a high level, and wasvery helpful in the study of exploring the effect of TORC1 in spinal cord injury.Partâ…¡The Biological Effects of Recombinant TORC1Lenti-virus Vector in vitroObjective To investigate the biological effects of recombinant TORC1 lenti-virusvector on spinal motor neuron (SMN) of SD rat. Methods SMNs were cultured usingnewborn SD rat of 1-3 d, and then transfected by recombinant TORC1 lenti-virus vector.The expression of TORC1 was detected by RT-PCR and Western Blot, and themorphological changes of SMN were observed as well. Results Recombinant TORC1lenti-virus vector could increase the expressions of TORC1 mRNA and protein in the SMN cells, and improve the growth of axons. Compared with normal SMNs and SMNstransfected by pGC-FU vector, the expressions of TORC1 mRNA and protein weresignificant higher (P＜0.05) and the length of axons was much more longer (P＜0.05) inthe experimental group. Conclusion The recombinant TORC1 lenti-virus vector couldeffectively increase the expression of TORC1 gene in SMNs and better the growth ofSMNs, which laid a foundation for further study.Partâ…¢The Biological Effects of RecombinantTORC1 Lenti-virus Vector in vivoObjective To investigate the biological effects of recombinant TORC1 lenti-virus vectoron the animal models of spinal cord injury. Methods Healthy adult mice were selectedand their spine were cut at T₁₀ level to establish the spinal cord injury models. Therecombinant TORC1 lenti-virus vector and pGC-FU vector were injected into the traumaarea separately during the operation. BBB scale of models and the expression of TORC1mRNA and protein were evaluated at 2 w, 4 w, 6 w after operation and immunohistochemistryof trauma area was performed as well to observe the process of nerve fiberregeneration. Results At 2 w after operation, compared with the negative group usingpGC-FU vector, the expression of TORC1 protein was up-regulated in the experimentalgroup(P＜0.05), while there was no difference in the expression of TORC1 mRNA, BBBscale and the number of regenerated nerve fiber in trauma area between the two groups(P＞0.05). However, at 4 w after operation, the experimental group took priority over thenegative control on all the 4 points mentioned above(P＜0.05).And things were the same at6 w after operation. Conclusion The recombinant TORC1 lenti-virus vector couldeffectively improve the repair of spinal cord injury in a long time, which made it apromising method to be applied clinically.